The long term goal of this proposal is to understand how a light- sensing organelle, the eyespot, is asymmetrically positioned in an algal cell and how it is assembled from chloroplast and host cell components at every cell division. The eyespot allows the unicellular alga to swim toward/away from light to optimize photosynthetic capacity. The eyespot is closely associated with a specific one of four highly acetylated microtubule rootlets that emanate from the region of the basal bodies. The eyespot is a complex sandwich of plasma membrane, outer and inner chloroplast membranes, carotenoid granules in the stroma of the chloroplast and thylakoid membranes. At cytokinesis, the eyespot is resorbed and two new eyespots are generated. It is a mystery how the eyespot is positioned, and how it is built de novo at each cell division. A genetic approach has identified six genes required for eyespot assembly and positioning. Mutations in four genes (EYE1-4) render the cells eyeless. Mutations in the MLT1 gene result in multiple eyespots that are incorrectly positioned. Mutations in the MIN1 gene result in smaller than normal eyespots and the close apposition of the plasma membrane and chloroplast envelope in the eyespot is lost. The EYE2 gene has been characterized and antisera raised to the protein. Insertion mutations that will allow isolation of the genes are in-hand for MIN1, MLT1 and EYE3. Genetics, molecular biology and immunofluorescence microscopy will be used to isolate genes and prepare fluorescent antibodies/proteins to probe the timing, placement and assembly of eyespot components during de novo eyespot synthesis at cytokinesis. Understanding cell polarity and the complex interaction of cells with their organelles is important. Disruption of cytoskeletal elements can lead to serious defects in tissue development and function. Organelle assembly and integration with the host cell structure and metabolism is essential for the health of cells, tissues and organisms. Fundamental lessons of biology learned through the power of genetics in model organisms will be the foundation for many advancements in human health.